H-1-H-2 EXCHANGE IN THE PERFUSED-RAT-LIVER METABOLIZING [3-C-13]ALANINE AND (H2O)-H-2 AS DETECTED BY MULTINUCLEAR NMR-SPECTROSCOPY

The exchange of individual protons of hepatic metabolites against the solvent deuterons has been investigated in perfused rat liver. Livers from starved rats were perfused for 20 min with a 10 mM solution of un labeled or 3-C-13-labeled L-alanine in Krebs Ringer bicarbonate buffer , with or without 50% deuterium oxide ((H2O)-H-2). High resolution C-1 3 NMR analysis of deuterium-induced isotopic shifts and of H-2-C-13 co uplings revealed a differential H-1-H-2 exchange depending on the chem ical nature of the metabolite and on the site of C-13 labeling. [3-C-1 3]Aspartate isotopomers showed similar H-2/H-1 ratios in the C3 and in the C2 carbons while [2-C-13]aspartate isotopomers had much smaller H -2/H-1 ratios in the C2 than in the C3 carbons. Similarly, [2-C-13]glu tamate isotopomers had H-2/H-1 ratios significantly smaller in the C2 than in the C3 carbon. These results suggest that the hydration-dehydr ation reactions of the citric acid cycle, which result in exchange at the C3 carbons of aspartate and glutamate, approach equilibrium with t he perfusate faster than the aminotransferases of aspartate and alanin e, which induce exchange at the C2 carbons of these amino acids. Taken together, the results obtained are consistent with a heterogeneous so lvent exchange environment in the perfused liver.